Protective immunity to pathogenic infection requires a complex set of effector responses mediated by multiple cell types. Effector CD4+ T cells are critical producers of IFN-gamma during infections by intracellular bacteria and protozoa (Th1 cells) and of IL-4 and IL-5 in response to parasites (Th2 cells). While much is known about the environmental effects of cytokines on T helper cell differentiation, the role of T cell intrinsic signaling pathways in this process is poorly understood. Tec-family tyrosine kinases are important regulators of phospholipase C-gamma activation following T cell antigen receptor stimulation. The goal of this proposal is to address the role of Tec kinases, Itk and Rlk, and Tec kinase-dependent signaling pathways, in Th1 vs. Th2 differentiation. Previous studies indicate that Itk-deficient mice are unable to mount protective immune responses to pathogens that require Th2 effector functions;in contrast Itk/Rlk-deficient mice regain this ability. Our recent findings provide insight into these data, by demonstrating that Itk signaling is required to inhibit T-bet induction following TCR stimulation;thus, in the absence of Itk, CD4+ T cells preferentially differentiate into Th1 effector cells. We have also found that Itk is upregulated in Th2 cells compared to Th1 cells, whereas Rlk is selectively lost from Th2 cells. On the basis of these data, we hypothesize that Itk and Rlk play distinct roles in regulating Th2 and Th1 differentiation and effector functions, respectively. We also hypothesize that Itk promotes Th2 differentiation via the negative regulation of T-bet expression. To address these hypotheses, we first propose to determine the mechanism by which Itk regulates T-bet, and to test the in vivo relevance of our findings using a T cell adoptive transfer lung airway inflammation (Th2) model. We will also determine whether constitutive Itk or Rlk expression in T cells interferes with, or biases, normal Th1 or Th2 differentiation, respectively. To determine the underlying basis for the paradoxically normal Th2 responses in Itk/Rlk-deficient mice, we will use a carefully controlled in vitro CD4+ T cell differentiation assay to examine the mechanisms regulating cytokine production and differentiation of Itk/Rlk-deficient cells. These studies will provide important information for future efforts to modulate Th1 vs. Th2 effector responses in vivo by manipulating distinct T cell signaling molecules and pathways.